molecules Article Capsaicin Causes Vasorelaxation of Rat Aorta through Blocking of L-type Ca2+ Channels and Activation of CB1 Receptors 1, 2, 3 Felipa Andrade y, Cinthia Rangel-Sandoval y, Alejandrina Rodríguez-Hernández , Evelyn López-Dyck 4, Alejandro Elizalde 2, Adolfo Virgen-Ortiz 2, Edgar Bonales-Alatorre 2 , Georgina Valencia-Cruz 2 and Enrique Sánchez-Pastor 2,* 1 National Technological Institute of Mexico/Technological Institute of Colima, Avenida Tecnológico No. 1, CP 28976 Villa de Álvarez, Colima, Mexico; [email protected] 2 University Center for Biomedical Research, University of Colima, Apdo. Postal No. 11, CP 28040 Colima, Colima, Mexico; [email protected] (C.R.-S.); [email protected] (A.E.); [email protected] (A.V.-O.); [email protected] (E.B.-A.); [email protected] (G.V.-C.) 3 Faculty of Medicine, University of Colima, Apdo. Postal No. 11, CP 28040 Colima, Colima, Mexico; [email protected] 4 State University of Sonora, Navojoa Academic Unit. Blvd. Manlio Fabio Beltrones 810, CP 85875 Navojoa, Sonora, Mexico; [email protected] * Correspondence: [email protected]; Tel.: +52-(312)-31-611-29 These authors contributed equally to this work. y Received: 1 June 2020; Accepted: 29 August 2020; Published: 30 August 2020 Abstract: The aim of this work was to determine whether Capsaicin may exert a vascular regulation through the activation of CB1 and/or CB2 receptors causing vasorelaxation in the rat aorta. Our results show the location of TRPV1 mainly in the endothelial and smooth muscle cells membrane. Nevertheless, Capsaicin caused vasorelaxation of this artery through a mechanism independent of TRPV1, since the specific antagonists Capsazepine and SB-366791 did not block the effect of Capsaicin. Because the significant expression of CB1 and CB2 receptors has been previously reported in the rat aorta, we used antagonists for these two receptors prior to the addition of Capsaicin. In these experiments, we found that the inhibition of CB1 using AM281, decreases the vasorelaxant effect caused by Capsaicin. On the other hand, the vasorelaxant effect is not altered in the presence of the CB2 receptor antagonist AM630. Furthermore, a partial decrease of the effect of Capsaicin was also seen when L-type calcium channels are blocked. A complete block of Capsaicin-induced vasorelaxation was achieved using a combination of Verapamil and AM281. In accordance to our results, Capsaicin-induced vasorelaxation of the rat aorta is neither dependent of TRPV1 or CB2 receptors, but rather it is strongly suggested that a tandem mechanism between inactivation of L-type calcium channels and the direct activation of CB1 receptors is involved. These findings are supported by CB1 docking simulation which predicted a binding site on CB1 receptors for Capsaicin. Keywords: cannabinoid receptor 1; TRPV1; Capsaicin; vasorelaxation 1. Introduction Blood pressure mainly depends on vascular tone, which is regulated by several mechanisms. These mechanisms include the release of some substances from the inner lining of the arteries such as NO, which causes dilation of the arteries or endothelin, which causes vasoconstriction [1]. Some substances cause vasorelaxation by interacting with ion channels in the medial layer or with some receptors such as Vanilloid, CB1, CB2, and/or GPR55 receptors. Molecules 2020, 25, 3957; doi:10.3390/molecules25173957 www.mdpi.com/journal/molecules Molecules 2020, 25, 3957 2 of 14 Vanilloid receptors are members of the Transient receptor potential channels (TRP channels) superfamily, which are non-selective cation channels and are found in different tissues, such as the skin, intestine, airways, and epithelial and endothelial cells [2]. TRPV1 is expressed in the smooth muscle and endothelium of the aorta and also in some other arteries, therefore, this receptor may have a very important role in the regulation of vascular tone [3]. These receptors can be activated by ligands such as capsaicinoids—one of these compounds is capsaicin (a component of hot peppers)—by endogenous ligands such as the endocannabinoid anandamide, and by other factors such as acidic pH (<5.3) and high temperatures (>43 ◦C) [2]. On the other hand, cannabinoid receptors (CB1 and/or CB2) are activated by endogenous cannabinoids (endocannabinoids) and exogenous cannabinoids, which are the active components of Cannabis sativa (marijuana). Both endocannabinoids and exogenous cannabinoids and synthetic cannabinoids cause vasorelaxation not only by interacting on cannabinoid receptors, but also by interacting with some other receptors (TRPV1, GPR55) or by their direct action on ion channels. Therefore, vanilloid and cannabinoid receptors may be related to the regulation of vascular tone, causing the vasorelaxation of some arteries, thus participating in the regulation of blood pressure [4–6]. The main TRPV1 agonist (Capsaicin) can cause vasorelaxation in several artery preparations as well. However, to date, the mechanisms of action of Capsaicin in modulating vascular tone is controversial. Although there are some reports implicating the role of TRPV1 in modulating vascular tone, some research has reported that Capsaicin causes vasorelaxation of rat aorta, rabbit coronary artery and human and porcine arteries by mechanisms independent of TRPV1 [5,7,8]. Therefore, the aim of the present work was to further investigate the different mechanisms involved in the effects of Capsaicin on the vascular tone of the rat aorta by determining the possible role of cannabinoid receptors in vasorelaxation caused by Capsaicin in the rat aorta. Here, we present evidence that Capsaicin induces vasorelaxation of the rat aorta by blocking L-type calcium channels and activating CB1, but not by activation of TRPV1 or CB2 receptors. 2. Results 2.1. Expression of TRPV1 Receptors in Smooth Muscle and Endothelial Cells of Rat Aorta Capsaicin is a known TRPV1 receptor agonist. These receptors are expressed in several rat arteries [9], however, their location in the smooth muscle or endothelial cell membrane where they can be activated and play a role in regulating vascular tone is unclear. Therefore, immunostaining followed by confocal microscopy experiments was used to evaluate the location of TRPV1 receptors in rat aortic rings. Thus, here we show the expression of TRPV1 receptors in both smooth muscle cells and endothelial cells of the rat aorta. Figure1 shows an aortic ring labeled with polyclonal rabbit anti-TRPV1 (Figure1A, green) and anti-smooth muscle α-actin (Figure1B, red). TRPV1 receptors are clearly located on smooth muscle cells as it can be seen as yellow color in the overlay (Figure1C). A dispersed smooth muscle cell of the aorta is shown in Figure1D, TRPV1 receptors (green) are expressed on the cell surface, as well as in the cytoplasm and nucleus (blue). Molecules 2020, 25, 3957 3 of 14 Molecules 2020, 25, x FOR PEER REVIEW 3 of 14 ABA B CDC D Figure 1. Expression of TRPV1 receptors in the smooth muscle of the rat aorta. (A) TRPV1 receptors Figure 1. Expression of TRPV1 receptors in the smooth muscle of the rat aorta. (A) TRPV1 receptors labeled with FITC (Green). The scale bar corresponds to 50 μm. (B) Aortic ring labeled with anti- labeled with FITC (Green). The scale bar corresponds to 50 µm. (B) Aortic ring labeled with anti-smooth smooth muscle α-actin conjugated with Alexa Fluor 568 (Red). (C) Overlay showing the high co- muscle α-actin conjugated with Alexa Fluor 568 (Red). (C) Overlay showing the high co-localization localization (Yellow) of TRPV1 receptors in smooth muscle. The arrows indicate the arterial layers. (Yellow) of TRPV1 receptors in smooth muscle. The arrows indicate the arterial layers. The adventitia The adventitia is marked with red, the tunica media with yellow and the tunica intima with blue. (D) is marked with red, the tunica media with yellow and the tunica intima with blue. (D) Aortic smooth Aortic smooth muscle cell labeled with anti-TRPV1 (Green) and stained with DAPI (Blue). The scale muscle cell labeled with anti-TRPV1 (Green) and stained with DAPI (Blue). The scale bar corresponds bar corresponds to 10 μm. to 10 µm. Furthermore,Furthermore, co-labeling of of the the endothelial endothelial cell cell laye layerr in an in aortic an aortic ring ringwith withpolyclonal polyclonal rabbit rabbitanti- anti-TRPV1TRPV1 (green) (green) and andmonoclonal monoclonal mous mousee anti-endothelial anti-endothelial cells cells(red) (red)is shown is shown on Figure on Figure 2. Thus,2. Thus, the location of these receptors on endothelial cells can be seen. Nuclei were stained using DAPI (Blue). the location of these receptors on endothelial cells can be seen. Nuclei were stained using DAPI (Blue). Our results corroborate the expression of TRPV1 receptors in both smooth muscle and endothelial Our results corroborate the expression of TRPV1 receptors in both smooth muscle and endothelial cells cells of the rat aorta and show evidence of their location in the membrane of these cells. of the rat aorta and show evidence of their location in the membrane of these cells. Molecules 2020, 25, 3957 4 of 14 Molecules 2020, 25, x FOR PEER REVIEW 4 of 14 ABC FigureFigure 2.2. ExpressionExpression ofof TRPV1TRPV1 receptors in rat aorta endothelium. (A (A) )TRPV1 TRPV1 was was detected detected by by a a specificspecific antibodyantibody andand aa FITC-conjugatedFITC-conjugated secondary secondary antibody antibody (Green). (Green). The The scale scale bar bar corresponds corresponds to 5 to μ 5 µm.m. (B (B).). The The tissue tissue was was co-labeled co-labeled with with an an endothe endothelium-specificlium-specific antibody antibody and and a asecondary secondary antibody antibody conjugatedconjugated with with AlexaAlexa FluorFluor 568; the cells are are locate locatedd along along the the inner inner layer, layer, in indicateddicated by by a ablue blue arrow. arrow. (C(C)) The The superimposition superimposition ofof thethe imagesimages highlightshighlights the co-localization of of the the receptor receptor in in the the endothelial endothelial cellscells (Arrowheads). (Arrowheads). NucleiNuclei werewere stainedstained using Prolong Gold antifade with with DAPI.